1. Field of the Invention
The present invention relates generally to the fields of biology and medicine. More particularly, it concerns a process for the ex vivo formation of bone and uses thereof.
2. Description of Related Art
The development of a functional tissue such as bone requires the concerted action of a number of microenvironmental signals: cytokines/growth factors, extracellular matrix (ECM) molecules, and cell:cell interactions. Moreover, these regulatory signals must be queued in the appropriate temporal and spatial order, resulting in a developmental microenvironment that facilitates three-dimensional growth. The skeletal system is no exception to such requirements. It is well understood that a number of cytokines/growth factors, such as TGF-xcex21 family members, modulate bone formation, and that ECM molecules like osteonectin, osteocalcin, and Type I and II collagen, etc., are important in both osteogenesis and chondrogenesis. As opposed to in vitro systems that are predominately planar, the need for three-dimensional tissue-like development is implicit both in the structural nature of the skeleton and its embryonic development. However, the extension of these in vivo spatial requirements to in vitro systems has been difficult and largely overlooked.
Cellular condensation, a process of cell aggregation mediated by mesenchymal:epithelial cell interactions, plays a crucial role during skeletogenesis (Hall and Miyake, 1992; 1995; Stringa et al., 1997). In the developing chick embryo, cellular condensation precedes differentiation into to prechondrocytes (Hall and Miyake, 1995). In contrast, during osteogenesis, cells differentiate to preosteoblasts and then undergo condensation (Hall and Miyake, 1995; Centrella, 1987). This condensation nonetheless precedes osteoblast differentiation and matrix mineralization (Dunlop and Hall, 1995). Studies of prechondrocytes demonstrate that cell condensation is cytokine-mediated, and induces changes in the expression of a number of developmentally important genes. For example, TGF-xcex21 or BMP2 both stimulate chondrocytic condensation and up-regulate fibronectin, N-CAM, and tenascin (Hall and Miyake, 1995). The requisite step of cellular condensation during mesenchymal chondrogenesis is mimicked in vitro in chondrocyte micromass cultures (Denker et al., 1995). Tuan and colleagues have demonstrated that TGF-xcex21 treatment of the multipotent C3H10T1/2 cells in small volumes of media at high cell density (i.e., micro-mass cultures) results in the formation of three dimensional structures that are cartilaginous in nature (Denker et a., 1995). These cellular condensations are associated with the up-regulation of cartilage extracellular matrix components such as Type II collagen and cartilage link protein (Denker et al., 1995). Likewise, studies of embryonic chick (calverial or limb-bud) cells confirm the cell-density mediated induction of chondrogenesis (Wong and Tuan, 1995; Woodward and Tuan, 1999), and demonstrate an obligate requirement for cell:cell interaction in this process, most likely mediated by N-cadherin (Woodward and Tuan, 1999), or N-CAM (Oberlender and Tuan, 1994; Miyake et al., 1996).
To date, no in vitro models of tissue-like osteogenic cell growth or cellular-condensation exist. Calverial or bone marrow-derived osteogenic cells are typically grown on two-dimensional (i.e., planar) surfaces. Cell proliferation eventually leads to a localized piling of confluent cells into xe2x80x9cbone nodules.xe2x80x9d This suggests that cell-density plays a role in the process of bone formation; however, studies directly demonstrating a relationship between cell-density and bone-formation are lacking, as are studies demonstrating the formation of three dimensional, crystalline bone (as opposed to reports concerning the mineralization of the extracellular matrix surrounding bone).
Present methods for the repair of bony defects include grafts of organic and synthetic construction. Three types of organic grafts are commonly used: autografts, allografts, and xenografts. An autograft is tissue transplanted from one site to another in the patient. The benefits of using the patient""s own tissue is that the graft will not evoke an immune response. However, using an autograft requires a second surgical site, which increases the risk of infection and may introduce additional complications. Further, bone available for grafting comes from a limited number of sites, for example, the fibula, ribs and iliac crest. An allograft is tissue taken from a different organism of the same species, and a xenograft from an organism of a different species. The latter types of tissue are readily available in larger quantities than autografts, but genetic differences between the donor and recipient may lead to rejection of the graft. All have advantages and disadvantages, yet none provides a perfect replacement for the missing bone.
There exists a need for a better way to repair and/or replace bone in subjects suffering from bone diseases or bone traumas.
The present invention concerns methods for the ex vivo formation of mammalian bone and subsequent uses of that bone. A critical and distinguishing feature of the present invention are defined tissue culture conditions and factors resulting in the formation of bone cell spheroids. xe2x80x9cBone cell spheroidsxe2x80x9d are defined as a tissue-like three dimensional growth of osteogenic cells or osteogenic precursor cells. The formation of bone cell spheroids permits the formation of bone within the spheroid. The invention also provides for methods of implanting the ex vivo formed bone into subjects. Also described are methods for genetically altering bone cells/spheroids to affect bone formation, identification of candidate modulators of bone formation, and identification of genes involved in bone formation.
Specifically, the present invention concerns a method for producing mammalian bone ex vivo by obtaining an osteogenic or bone precursor cell; culturing the cell under serum free conditions in the presence of one or more osteogenic growth factors; and establishing the cell cultures at cell densities that allow the formation of a bone cell spheroid containing bone, whereby bone is formed within cells of the bone cell spheroid. The osteogenic or bone precursor cell of the present invention can be isolated from primary sources such as bone marrow or bone explants. Protocols for isolating such cells are described herein. In other embodiments, cell lines of bone cell derivation can be utilized. Osteogenic or bone precursor cells can be from several mammalian species, including but not limited to, human, bovine, equine, canine, feline, chick, rat, or murine origin.
The present invention describes the culturing of the osteogenic cell in defined, serum-free media. Defined medias are described herein as well as additives that are commonly found in these defined medias, including albumin, insulin, an iron source, a fatty acid source and other essential components. The defined serum free media of the present invention also is supplemented with growth factors that are important for the formation of bone cell spheroids. The primary growth factors are broadly defined as members of the Transforming Growth Factor xcex2 (TGF-xcex2) gene superfamily. Members of this family include TGF-xcex21, TGF-xcex22, TGF-xcex21.2, and Bone Morphogenic Protein 2 (BMP-2), BMP-4 and BMP-7. Other growth factors such as parathyroid hormone (PTH), calcitonin, 1,25-dihydroxy vitamin D3, interleukin-6, insulin-like growth factors (IGFs) I and II, VEGF and interleukin-11 can be used as solitary or costimulatory factors.
The osteogenic or bone precursor cell of the present invention may be purified by physico-chemical separation techniques, such as equilibrium density separation. In other embodiments, the osteogenic or bone precursor cell may be purified by immuno-affinity isolation, such as those utilizing immune adhesion, immuno-column chromatography, or fluorescence-activated cell sorting. In preferred embodiments, the immuno-affinity isolation utilizes antibodies to osteocalcin, osteonectin, or alkaline phosphatase, or combinations thereof.
The osteogenic or bone precursor cell cultures of the present invention are initiated at cell-densities are from about 1.0xc3x97103 to about 1xc3x97106 cells per cm2.
Further embodiments of the invention describe a method of providing bone tissue to a mammal, comprising obtaining a bone cell spheroid and implanting the bone cell spheroid into a mammal. The bone cell spheroid may be implanted in a gel, including alginate gels, collagen gels, or fibrin gels. In other embodiments, the bone cell spheroid is implanted in polylactic acid, polyglycolic acid, or PGLA. The bone cell spheroid may also be implanted in or on hydroxyapatitic or other apatitic compounds, devitalized animal bone, devitalized human bone, or porous ceramic structures.
The present invention also concerns implanting a bone cell spheroid in conjunction with orthopedic surgery and/or orthopedic devices, such as hip implants, knee implants, and spinal fusions. Alternatively, implanting a bone cell spheroid is in conjunction with oral surgery and/or dental implants, plastic surgery, or periodontal repairs. Implantation of a bone cell spheroid may be into bone-forming tissue or into a wound. Implantation of a bone cell spheroid also may be into a mammal which has a bone disease such as osteoporosis, Vitamin D deficiency, Osteotitis deformans, Von Recklinghausen""s Disease.
The present invention also concerns a method for producing mammalian bone ex vivo by obtaining an osteogenic or bone precursor cell; culturing the cell under serum free conditions in the presence of one or more growth factors of the TGF-xcex2 gene superfamily; maintaining the cell cultures at cell densities that allow the formation of a bone cell spheroid, and bone therein; and removing the cellular elements allowing the use of the resulting bone in vivo.
Other embodiments of the invention concern a method for producing mammalian bone ex vivo by obtaining an osteogenic or bone precursor cell; culturing the cell under serum free conditions in the presence of one or more osteogenic growth factors; contacting said cell with a recombinant cDNA containing vector that directs the expression of a protein that enhances bone and/or bone cell spheroid formation; and maintaining the cell cultures at cell densities that allow the formation of a bone cell spheroid. The vector can be a plasmid or a viral vector. Methods for producing and delivering the vector to the osteogenic or bone precursor cell are described. Proteins that enhance bone cell spheroid formation that can be expressed by the cDNA""s include, but are not limited to, members of the TGF-xcex2 gene superfamily, including TGF-xcex21, TGF-xcex22, TGF-xcex21.2, BMP-2, BMP-4 and BMP-7, as well as cDNA""s encoding extracellular matrix proteins such as osteonectin, osteopontin, osteocalcin, bone sialoprotein, collagen, fibronectin, thrombospondin, insulin-like growth factors I or II, or VEGF. Also contemplated are cDNA""s directing the expression of cytoadhesion molecules such as integrins, selectins and cadherins, or growth factors such as PTH, calcitonin, interleukin-6 or interleukin-11.
Further embodiments of the invention describe a method for using mammalian bone for bone repair in a subject by obtaining an osteogenic or bone precursor cell; culturing the cell under serum free conditions in the presence of one or more osteogenic growth factors; contacting said cell with a recombinant cDNA containing vector that directs the expression of a protein that enhances bone cell spheroid formation; and initiating the cell cultures at cell densities that allow the formation of a bone cell spheroid; removing the cellular elements from the ex vivo formed bone; and using the formed bone to effect repair.
Another embodiment of the present invention concerns a method for identifying a gene involved in mammalian bone formation, bone repair and/or bone disease by obtaining an osteogenic or bone precursor cell; culturing the cell under serum free conditions in the presence of one or more osteogenic growth factors; initiating the cell cultures at cell-densities that allow the formation of a bone cell spheroid; and identifying genes that are expressed during or following the formation of a bone cell spheroid and not expressed in osteogenic or bone precursor cells cultured in control culture conditions. Methods for identifying genes include differential message display assays, polymerase chain reaction based assays, Northern analysis and gene expression arrays (e.g., cDNA-based or oligonuleotide-based gene chips).
The present invention also concerns a method for producing a modulator of mammalian bone formation, bone repair and/or bone disease by the steps of obtaining an osteogenic or bone precursor cell; culturing the cell under serum free conditions in the presence of a candidate modulator, but in the absence of other osteogenic growth factors; measuring bone cell spheroid formation; and comparing the formation of bone and/or bone cell spheroid with that observed in the presence of one or more osteogenic growth factors. In another embodiment, the present invention concerns a method for identifying a modulator of mammalian bone formation, bone repair or bone disease by obtaining an osteogenic or bone precursor cell; culturing said cell under serum free conditions in the presence of a candidate modulator plus the presence of one or more growth factors of the TGF-xcex2 gene superfamily; measuring bone cell spheroid formation; comparing the formation of bone cell spheroid with that observed in the absence of the modulator, and producing a modulator so identified. Modulators identified by this assay would be expected to enhance, inhibit, or act synergistically with growth factors of the TGF-xcex2 gene superfamily. Currently, the best example of this latter concept is shown by co-stimulation of spheroid formation with TGFB1 and PTH which increases the size of the resulting microspicules.
The present invention also describes a bone cell spheroid made by the process of obtaining an osteogenic cell or bone precursor cell; culturing said cell under serum free conditions in the presence of one or more osteogenic growth factors; and maintaining the cell cultures at cell densities that allow the formation of a bone cell spheroid.